Structure and function of PA4872 from Pseudomonas aeruginosa, a novel class of oxaloacetate decarboxylase from the PEP mutase/isocitrate lyase superfamily

Abstract

Pseudomonas aeruginosa PA4872 was identified by sequence analysis as a structurally and functionally novel member of the PEP mutase/isocitrate lyase superfamily and therefore targeted for investigation. Substrate screens ruled out overlap with known catalytic functions of superfamily members. The crystal structure of PA4872 in complex with oxalate (a stable analogue of the shared family alpha-oxyanion carboxylate intermediate/transition state) and Mg2+ was determined at 1.9 A resolution. As with other PEP mutase/isocitrate lyase superfamily members, the protein assembles into a dimer of dimers with each subunit adopting an alpha/beta barrel fold and two subunits swapping their barrel's C-terminal alpha-helices. Mg2+ and oxalate bind in the same manner as observed with other superfamily members. The active site gating loop, known to play a catalytic role in the PEP mutase and lyase branches of the superfamily, adopts an open conformation. The Nepsilon of His235, an invariant residue in the PA4872 sequence family, is oriented toward a C(2) oxygen of oxalate analogous to the C(3) of a pyruvyl moiety. Deuterium exchange into alpha-oxocarboxylate-containing compounds was confirmed by 1H NMR spectroscopy. Having ruled out known activities, the involvement of a pyruvate enolate intermediate suggested a decarboxylase activity of an alpha-oxocarboxylate substrate. Enzymatic assays led to the discovery that PA4872 decarboxylates oxaloacetate (kcat = 7500 s(-1) and Km = 2.2 mM) and 3-methyloxaloacetate (kcat = 250 s(-1) and Km = 0.63 mM). Genome context of the fourteen sequence family members indicates that the enzyme is used by select group of Gram-negative bacteria to maintain cellular concentrations of bicarbonate and pyruvate; however the decarboxylation activity cannot be attributed to a pathway common to the various bacterial species.

Figure 1

The reactions catalyzed by the PEP mutase/isocitrate lyase superfamily. The reactions are depicted to occur stepwise with the formation of reaction intermediates (shown in brackets). The reactions might be concerted in which case the transition state will resemble the intermediate.

Figure 2

Overall fold of PA4872. (A) The tetramer structure highlighting each subunit in different color. Mg²⁺ is depicted as yellow sphere and oxalate as stick models with an atomic coloring scheme: Carbon - green, oxygen – red. (B) Superposition of the PA4872 (gold and salmon) and 2-methylisocitrate lyase (purple) monomers, illustrating the different paths of the C-terminal chains.

Figure 3

Active site of PA4871. (A) The electron density associated with key active site residues. The sigma(a)-weighted 2F_o – F_c map is shown contoured at 2σ level. Atomic colors are used: Carbon – green, oxygen – red, nitrogen – blue, Mg²⁺ - yellow. Oxalate is labeled Oxl (B) stereoscopic representation of the active site. Atomic colors are: Carbon – gray, oxygen – red, nitrogen – blue, Mg²⁺ - green.

Figure 4

Structure based sequence alignment of PA4872 close relatives. Invariant active site residues that are conserved also in other PEP mutase/isocitrate lyase superfamily members are highlighted in blocked green. Other invariant residues are highlighted in blocked gray except for the active site His235, Tyr 212 (and the nearby glutamate of the 3-protein subgroup) and Ser27, which are highlighted in blocked red. Secondary structure motifs are shown above the sequences.

Figure 5

Molecular surfaces in the vicinity of active sites. (A) The channel leading to the active site of PA4872 (indicated by an arrow). Mg²⁺-oxalate, His235 and Tyr212 are shown for reference. (B) The surface around the active site of 2-methylisocitrate lyase corresponding to the Mg²⁺-pyruvate where the gating loop is in the open conformation (1).

Figure 6

The type of intermediates formed by the mutase/transferase and lyase branches of the superfamily.

Figure 7

The active site cavity (shown as a cage) with docked potential substrates shown as stick models with the atomic colors green – carbon, red – oxygen. The Mg²⁺ is depicted as a magenta sphere.

Figure 8

Proposed catalytic mechanism of PA4872 and comparison with the catalytic machinery of the malic enzyme oxaloacetate decarboxylase.

Figure 9

pH profile of PA4872. The buffers used in various pH ranges are listed in the Methods section.